FAP directed target modules are suitable for imaging and targeted radionuclide therapy of FAP-expressing solid tumours and their microenvironment

Fibroblast activation protein (FAP), mainly expressed by cancer-associated fibroblasts (CAFs) in the tumour stroma, promotes tumour growth, metastasis, and immunosuppression and, therefore, has been studied as a target for cancer diagnosis and treatment. With regard to immunotherapy, the innovative modular universal CAR (UniCAR) platform developed by our group is one of the most promising approach due to the reduced risk for e.g. on-target/off-tumour toxicities and cytokine release syndrome. Thereby, chimeric antigen receptor (CAR) T-cells (UniCAR T cells) are exclusively activated in the presence of a target module (TM) that specifically establishes the crosslinking between target cells and UniCAR T-cells. FAP specific TMs are hypothesized to be not only immunotherapeutics with increased safety but in addition to be suitable as radionuclide-based theranostic agents.
For that, low molecular weight TMs that are rapidly eliminated allowing a specific and recurrent on/off switch of UniCAR T-cell activity via TM dosing were developed by fusion of the single-chain variable fragment (scFv) of an anti-human FAP mAb to the peptide epitope E5B9 that is recognized by the UniCAR T-cells. To ease the clinical TM administration at later stages of tumour therapy and for targeted radionuclide therapy, however, TMs with extended half-life may be advantageous. Therefore, anti-FAP TMs based on the human IgG4 Fc-domain, including a mutated version, were created. All TMs were tested (i) in vitro based on naturally and artificially overexpressing 2D and 3D models and (ii) in vivo by positron emission tomography (PET) and single-photon emission tomography (SPECT) in NMRI nude mice bearing both mock transfected and FAP overexpressing HT1080 tumor xenografts.
In vitro, all TMs were proven to specifically redirect UniCAR T-cells to FAP-expressing target cells. Moreover, FAP specific TMs could be conjugated to different chelators, e.g. Bispidines, NODAGA, and CHX-A-DTPA and, afterwards, radiolabelled with either Copper-64 or Lutetium-177. PET imaging with 64Cu radiolabelled anti-FAP IgG4 TMs revealed an excellent FAP specific tracer enrichment at the tumour site already 6h p.i. After 24 to 48h p.i. tumor SUVmean increased up to 20 with almost no background. SPECT imaging with 177Lu radiolabelled anti-FAP IgG4 TMs confirmed the high FAP-dependent tumour uptake and, thereby, offers possibility for targeted radionuclide therapy.
In conclusion, we designed novel FAP specific TMs with different molecular weight that can be used for immunotherapeutic approaches using UniCAR T-cells, diagnostic imaging, and targeted radionuclide therapy and, thereby, have the potential to improve cancer treatment allowing an individualized treatment of cancer patients with increased clinical safety.